Whitening composition including novel kaempferol-based compound derived from post-fermented tea

ABSTRACT

The present specification relates to a whitening composition including a novel compound isolated from a post-fermented tea, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof, and may be widely used in various areas related to skin whitening and skin care.

TECHNICAL FIELD

The present specification relates to a whitening composition including anovel kaempferol-based compound.

BACKGROUND ART

Recently, consumers' interest in whitening is increasing due to theincrease in ultraviolet rays caused by environmental pollution ordestruction of the ozone layer. However, as a great deal of side effectsand hypersensitivity reactions to chemical synthetic cosmetics preparedwith artificial compounds have been reported, attempts have beenactively made to obtain raw materials for cosmetics from naturalsubstances. Kojic acid, arbutin or its derivatives, which have beenidentified to date, have not been sufficiently used as raw materials forcosmetics because they have deficiencies in safety or stability or theirwhitening effect is not sufficient when actually mixed as raw materialsfor cosmetics. Accordingly, studies to find the whitening activeingredients in natural substances have been continuously conducted, andmaterials that show the inhibition of tyrosinase activity have beenfound. However, there still remains an issue to be solved, such asissues of stability or appropriate effective concentration.

On the other hand, green tea is drinkable in the form of leaf tea, orfermented tea for a deeper flavor. Fermented green tea means that thegreen tea leaves are subjected to oxidation treatment, and includesfermented tea oxidized by the oxidase present in the tea leaves, andpost-fermented tea fermented by a separate microorganism other than theenzyme present in the tea leaves. Depending on the degree offermentation, it can be classified into weakly fermented tea,semi-fermented tea, and fully fermented tea. For example, fermentedgreen tea is called by various names, such as green tea, Oolong tea,black tea, puer tea, etc., depending on the type and extent offermentation.

The fermented tea may not only show a difference in flavor compared toleaf tea, but may also show a big difference in the type and content ofactive ingredients depending on the specific fermentation process andthe type of microorganism. Since various compounds can be produced andseparated, various efforts for separating and identifying unknown novelcompounds using green tea have been continued.

CITATION LIST Patent Literature

[Patent Literature 1] Korean Patent No. 10-0975199

SUMMARY OF INVENTION Technical Problem

An object of the present disclosure is to provide a novel compoundderived from post-fermented tea for whitening use.

Solution to Problem

In one embodiment, the present disclosure provides a whiteningcomposition including a compound of Formula 1, an optical isomerthereof, a pharmaceutically acceptable salt thereof, a hydrate thereof,a solvate thereof, or a post-fermented tea extract including the same asan active ingredient.

In Formula 1 above, R₁ may be C₁₅H₉O₆, R₂ may be C₆H₁₁O₅, and R₃ may beC₉H₇O₂.

In another aspect of the present disclosure, the composition may be acomposition that inhibits one or more of tyrosinase activity and melaninproduction.

In one embodiment, the present disclosure provides a method for skinwhitening comprising administering a compound of Formula 1, an opticalisomer thereof, a pharmaceutically acceptable salt thereof, a hydratethereof, a solvate thereof, or a post-fermented tea extract comprisingthe same to a subject in need thereof.

In one embodiment, the present disclosure provides a use of a compoundof Formula 1, an optical isomer thereof, a pharmaceutically acceptablesalt thereof, a hydrate thereof, a solvate thereof, or a post-fermentedtea extract comprising the same in manufacture of a composition for skinwhitening.

In one embodiment, the present disclosure may provide a compound ofFormula 1, an optical isomer thereof, a pharmaceutically acceptable saltthereof, a hydrate thereof, a solvate thereof, or a post-fermented teaextract comprising the same for use in skin whitening.

Advantageous Effects of Invention

In one aspect, the present disclosure can be widely used inpost-fermented tea-related industries, skin care related fields, or thelike, by allowing novel compounds isolated from post-fermented tea to beused in whitening fields.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an MS spectrum of the compound according to an aspect ofthe invention.

FIG. 2 shows a ¹H-NMR (nuclear magnetic resonance) spectrum of thecompound according to an aspect of the present disclosure.

FIG. 3 shows a ¹³C-NMR spectrum of the compound according to an aspectof the present disclosure.

FIG. 4 shows an ¹H-¹³C HSQC (Heteronuclear Single Quantum Coherence)spectrum of the compound according to an aspect of the presentdisclosure.

FIG. 5 shows a ¹H-¹³C HMBC (Heteronuclear Multiple-Bond Coherence)spectrum of the compound according to an aspect of the presentdisclosure.

DESCRIPTION OF EMBODIMENTS

Hereinafter, the present disclosure will be described in detail.

As used herein, “post-fermentation” includes fermentation by a separatemicroorganism or substance other than the enzyme present in tea leaves.Post-fermented tea includes green tea fermented by the above method.

As used herein, the term “extract” means a substance obtained byextracting a component contained inside of a natural substance,regardless of the extracted method or ingredients. The term is used in abroad sense including, for example, extracting a component soluble in asolvent from a natural substance using water or an organic solvent,extracting only a specific component of a natural substance such as oil,and fraction that fractionated those thus obtained again by using aspecific solvent or the like.

As used herein, “fractions” include those obtained by fractionating aspecific substance or extract using a certain solvent or those leftafter fractionating, and extracting them again with a specific solvent.Fractional methods and extraction methods may be any method known tothose skilled in the art.

As used herein, “isomers” include, in particular, not only opticalisomers (e.g., essentially pure enantiomers, essentially purediastereomers or mixtures thereof), but also conformation isomers (i.e.,isomers that differ only in their angles of one or more chemical bonds),position isomers (especially tautomers) or geometric isomers (e.g.,cis-trans isomers).

As used herein, the term “essentially pure” means that a specificcompound, for example enantiomers or diastereomers, when used inconnection with an enantiomer or diastereomer, is present in an amountof at least about 90%, preferably at least about 95%, more preferably atleast about 97% or at least about 98%, even more preferably at leastabout 99%, and even more further preferably at least about 99.5% (w/w).

As used herein, the term “pharmaceutically acceptable” refers to thosethat can be approved or approved by the government or equivalentregulatory agencies for use in animals, more specifically in humans, byavoiding significant toxic effects when used in conventional medicinaldosage, or those recognized as being listed in the pharmacopoeia ordescribed in other general pharmacopoeia.

As used herein, the term “pharmaceutically acceptable salt” refers to asalt according to one aspect of the present disclosure that ispharmaceutically acceptable and possesses the desired pharmacologicalactivity of the parent compound. The salts include (1) acid additionsalts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, or the like; or (2)salts formed when an acidic proton present in the parent compound issubstituted.

As used herein, the term “hydrate” refers to a compound to which wateris bound, and is a broad concept including an inclusion compound havingno chemical bonding force between water and the compound.

As used herein, the term “solvate” refers to a compound of higher orderproduced between molecules or ions of a solute and molecules or ions ofa solvent.

In one aspect, the present disclosure provides a composition for skinwhitening including a compound of following Formula 1, an optical isomerthereof, a pharmaceutically acceptable salt thereof, a hydrate thereof,a solvate thereof, or a post-fermented tea extract comprising the sameas an active ingredient.

In Formula 1 above, R₁ may be C₁₅H₉O₆, R₂ may be C₆H₁₁O₅, and R₃ may beC₉H₇O₂.

In one embodiment, R₁ may be a compound of following Formula 2.

In another embodiment, R₂ may be a compound of following Formula 3.

R₃ may be a compound of following Formula 4.

In another embodiment, the compound may bekaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside]).The compound may be represented by the following Formula 5.

According to one aspect of the present disclosure, a method forpreparing the compounds, isomers thereof, pharmaceutically acceptablesalts thereof, hydrates thereof or solvates thereof may includesynthesis, separation from natural substances, or the like.

According to another embodiment, the post fermentation may be by straininoculation, and the strain may be selected from Saccharomyces sp.,Bacillus sp., Lactobacillus sp., and Leuconostoc mesenteroides sp., andmay preferably be selected from Saccharomyces cerevisiae, Lactobacilluscasei, Bacillus subtlis, Lactobacillus bulgarius and Leuconostocmesenteroides. According to another embodiment, the post-fermented teamay be post-fermented green tea.

In one aspect of the present disclosure, the compound is a compounddiscovered by the present inventors after the continuous studies of thepost-fermented tea, and it was confirmed that the compound is effectivein inhibiting one or more of tyrosinase activity and melanin production.Accordingly, it has been demonstrated that the compound can be used forthe purpose of skin whitening or skin care using the compound accordingto one aspect of the present disclosure (see Examples 1 and 2).

Melanin is found in the outer feathers, skin, head, eyes, etc. ofanimals. When melanin is overproduced, it is deposited on the skin toform spots and freckles, promote skin aging, and may even cause skincancer. The diseases or symptoms caused by overproduction of melanin maybe one or more selected from the group consisting of spots, freckles,age spots, blemishes, epidermal melanocytic lesion, cafe's au laitmacules, birthmarks, Becker's nevus, nevus spilus, lentigines, lentigos,dermal melanocytic lesions, mongolian spot, nevus of Ota, acquiredbilateral nevus of Ota-like macules, nevus of Ito, blue nevus,melanocytic nevus, junctional nevus, compound nevus, intradermal nevus,halo nevus, congenital nevocytic nevus, Spitz nevus, dysplastic nevus,melanoma, lentigo maligna melanoma, superficial spreading melanoma,acral lentiginous melanoma, nodular melanoma, pigment basal cellcarcinoma, dermatofibromas, dermoid cyst, keloid, ultravioletradiation-induced pigmentation (especially melanin), drug-inducedpigmentation (especially melanin), pigmentation following inflammation(especially melanin), pigmentation caused by dermatitis (especiallymelanin) and keratoacanthomas.

Since the compound according to one aspect of the present disclosure iseffective in inhibiting one or more of tyrosinase activity and melaninproduction, the compound may also be used for the purpose of preventing,treating and improving diseases or symptoms caused by the melanindeposition and overproduction (see Experimental Examples 1 and 2).

In one embodiment, the extraction may be extraction by one or moresolvents selected from water, hydrothermal water, lower alcohols of C₁to C₆, and mixed solvents thereof, and in another embodiment, the loweralcohol may be an alcohol alone or a mixture which can be generally usedin the art, and may preferably be ethanol.

According to another aspect of the present disclosure, the extract maybe a fraction fractionated with ketones after extraction.

In another embodiment, examples of the ketones include acetone, carvon,pulegone, isolongifolanone, 2-heptanone, 2-pentanone, 3-hexanone,3-heptanone, 4-heptanone, 2-octanone, 3-octanone, 2-nonanone,3-nonanone, 2-undecanone, 2-tridecanone, methyl isopropyl ketone, ethylisoamyl ketone, butylidene acetone, methylheptenone, dimethyl octenone,geranyl acetone, farnesyl acetone, 2,3-pentadione, 2,3-hexadione,3,4-hexadione, 2,3-heptadione, amylcyclopentanone, amylcyclopentenone,2-cyclopentyl cyclopentanone, hexylcyclopentanone,2-n-heptylcyclopentanone, cis-jasmon, dihydrojasmon, methylcorylone,2-tert-butylcyclohexanone, p-tert-butylcyclohexanone,2-sec-butylcyclohexanone, celery ketone, krypton,p-tert-pentylcyclohexanone, methylcyclocitron, neron,4-cyclohexyl-4-methyl-2-pentanone, oxide ketone, emoxyprone,methylnaphthyl ketone, α-methylanisal acetone, anisyl acetone, p-methoxyphenyl acetone, benzylidene acetone, p-methoxyacetophenone,p-methylacetophenone, propiophenone, acetophenone, α-dynascone, lritone,ionone, pseudoionone, methylionone, methyl lritone,2,4-di-tert-butylcyclohexanone, allylionone,2-acetyl-3,3-dimethylnorbornane, verbenone, fenchon, cyclopentadecanone,cyclohexadecenone, or the like, may include both ketones and mixturesthereof as solvents that can be generally used in the art, and maypreferably be acetone.

According to one aspect of the present disclosure, the content of thecompound of Formula 1, an isomer thereof, a pharmaceutically acceptablesalt thereof, a hydrate thereof, or a solvate thereof in the compositionmay be 0.00001% to 10% by weight based on the total weight of thecomposition. The content may be at least 0.00001% by weight, at least0.00005% by weight, at least 0.0001% by weight, at least 0.0005% byweight, at least 0.001% by weight, at least 0.005% by weight, at least0.01% by weight, at least 0.05% by weight, at least 0.1% by weight, atleast 0.5% by weight, at least 1% by weight, at least 2% by weight, atleast 3% by weight, at least 4% by weight, at least 5% by weight, atleast 6% by weight, at least 7% by weight, at least 8% by weight, or atleast 9% by weight. In addition, the content may be 10% or less byweight, 9% or less by weight, 8% or less by weight, 7% or less byweight, 6% or less by weight, 5% or less by weight, 4% or less byweight, 3% or less by weight, 2% or less by weight, 1% or less byweight, 0.5% or less by weight, 0.1% or less by weight, 0.05% or less byweight, 0.01% or less by weight, 0.005% or less by weight, 0.001% orless by weight, 0.0005% or less by weight, 0.0001% or less by weight,0.00005% or less by weight, or 0.00003% or less by weight.

According to another aspect of the present disclosure, the content ofthe post-fermented tea extract in the composition may be 0.1% to 90% byweight based on the total weight of the composition. The content may beat least 0.1% by weight, at least 1% by weight, at least 5% by weight,at least 10% by weight, at least 15% by weight, at least 20% by weight,at least 25% by weight, at least 30% by weight, at least 35% by weight,at least 40% by weight, at least 45% by weight, at least 50% by weight,at least 55% by weight, at least 60% by weight, at least 65% by weight,at least 70% by weight, at least 75% by weight, at least 80% by weight,or at least 85% by weight based on the total weight of the composition.In addition, the content may be 90% or less by weight, 85% or less byweight, 80% or less by weight, 75% or less by weight, 70% or less byweight, 65% or less by weight, 60% or less by weight, 55% or less byweight, 50% or less by weight, 45% or less by weight, 40% or less byweight, 35% or less by weight, 30% or less by weight, 25% or less byweight, 20% or less by weight, 15% or less by weight, 10% or less byweight, 5% or less by weight, 1% or less by weight, or 0.5% or less byweight.

According to another aspect of the present disclosure, the extract maycontain the compound of Formula 1, an isomer thereof, a pharmaceuticallyacceptable salt thereof, a hydrate thereof, or a solvate thereof in anamount of at least 0.00001% by weight, at least 0.00005% by weight, atleast 0.0001% by weight, at least 0.0005% by weight, at least 0.001% byweight, at least 0.005% by weight, at least 0.01% by weight, at least0.05% by weight, at least 0.1% by weight, at least 0.5% by weight, atleast 1% by weight, at least 3% by weight, at least 5% by weight, atleast 7% by weight, at least 10% by weight, at least 12% by weight, atleast 15% by weight, or at least 18% by weight based on the total weightof the extract. In addition, the content may be 20% or less by weight,15% or less by weight, 12% or less by weight, 10% or less by weight, 7%or less by weight, 5% or less by weight, 3% or less by weight, 1% orless by weight, 0.5% or less by weight, 0.1% or less by weight, 0.05% orless by weight, 0.01% or less by weight, 0.005% or less by weight,0.001% or less by weight, 0.0005% or less by weight, 0.0003% or less byweight, 0.00005% or less by weight, or 0.00003% or less by weight.Preferably, the extract may contain 0.00001% to 20% by weight of thecompound of Formula 1, an isomer thereof, a pharmaceutically acceptablesalt thereof, a hydrate thereof, or a solvate thereof based on the totalweight of the extract.

According to another aspect of the present disclosure, the dosage of thecompound of Formula 1, an isomer thereof, a pharmaceutically acceptablesalt thereof, a hydrate thereof, or a solvate thereof by administrationof the composition may be 0.00001 mg/kg/day to 100 mg/kg/day. The dosagemay be at least 0.00001 mg/kg/day, at least 0.0001 mg/kg/day, at least0.001 mg/kg/day, at least 0.005 mg/kg/day, at least 0.01 mg/kg/day, 0.05mg/kg/day, at least 0.1 mg/kg/day, at least 0.5 mg/kg/day, at least 1mg/kg/day, at least 5 mg/kg/day, at least 10 mg/kg/day, at least 15mg/kg/day, at least 20 mg/kg/day, at least 25 mg/kg/day, at least 30mg/kg/day, at least 35 mg/kg/day, at least 40 mg/kg/day, at least 45mg/kg/day, at least 50 mg/kg/day, at least 55 mg/kg/day, at least 60mg/kg/day, at least 65 mg/kg/day, at least 7 mg/kg/day, at least 75mg/kg/day, at least 80 mg/kg/day, at least 85 mg/kg/day, at least 90mg/kg/day, or at least 95 mg/kg/day. In addition, the dosage may be 100mg/kg/day or less, 95 mg/kg/day or less, 90 mg/kg/day or less, 85mg/kg/day or less, 80 mg/kg/day or less, 75 mg/kg/day or less, 70mg/kg/day or less, 65 mg/kg/day or less, 60 mg/kg/day or less, 55mg/kg/day or less, 50 mg/kg/day or less, 45 mg/kg/day or less, 40mg/kg/day or less, 35 mg/kg/day or less, 30 mg/kg/day or less, 25mg/kg/day or less, 20 mg/kg/day or less, 15 mg/kg/day or less, 10mg/kg/day or less, 5 mg/kg/day or less, 1 mg/kg/day or less, 0.5mg/kg/day or less, 0.1 mg/kg/day or less, 0.05 mg/kg/day or less, 0.01mg/kg/day or less, 0.005 mg/kg/day or less, 0.003 mg/kg/day or less,0.001 mg/kg/day or less, 0.0005 mg/kg/day or less, 0.0001 mg/kg/day orless, 0.00005 mg/kg/day or less.

According to another aspect of the invention, the composition may be afood composition, a cosmetic composition, or a pharmaceuticalcomposition.

Food composition according to one aspect of the present disclosure maybe a health food composition. In the health food composition, the dosagedetermination of the compound is within the level of those skilled inthe art, and may vary depending on a variety of factors including theage, health condition and complications, or the like of the subject tobe administered.

The health food composition according to one aspect of the presentdisclosure may be a health functional food, as well as, for example, maybe any form of processed food including various foodstuffs such aschewing gum, caramel product, candy, ice cream, confectionery, breads,or the like, beverages such as soft drinks, mineral water, alcoholicbeverages, or the like, and may be functional foodstuffs includingvitamins, minerals, or the like.

In addition to the above, the health food composition according to oneaspect of the present disclosure may contain various nutrients,vitamins, minerals (electrolytes), flavors such as synthetic flavoringsand natural flavorings, colorants and enhancers (cheese, chocolate, orthe like), pectic acid and salts thereof, alginic acid and saltsthereof, organic acids, protective colloid thickeners, pH adjustingagents, stabilizers, preservatives, glycerin, alcohols, and carbonatingagents used in carbonated beverages. In addition, the functional foodcompositions of the present disclosure may contain natural fruit juiceand flesh for the production of fruit juice drinks and vegetable drinks.These components may be used independently or in combination. Theproportion of such additives is not so critical, but the additives aregenerally contained in the range of from 0 to about 50 parts by weightper 100 parts by weight of the composition according to one aspect ofthe present disclosure.

The cosmetic composition according to one aspect of the presentdisclosure is a composition for skin, nails and/or hair, and may have aformulation, for example, softening tonic, astringent tonic, nourishingtonic, nourishing cream, massage cream, eye cream, eye essence, essence,cleansing cream, cleansing lotion, cleansing foam, cleansing water,pack, powder, body lotion, body cream, body essence, body cleanser, hairdye, shampoo, rinse, hair conditioner, hair tonic, ointment, gel, cream,patch, spray and skin adhesive type, or the like, but is not limitedthereto.

In addition, in each formulation, other ingredients in addition to theabove essential ingredients may be appropriately selected and blended bythose skilled in the art without difficulties according to the kind orpurpose of use of other external preparations.

The cosmetic composition may be provided in any formulation suitable fortopical application. For example, it may be provided in forms of asolution, an emulsion obtained by dispersing an oil phase in an aqueousphase, an emulsion obtained by dispersing an aqueous phase in an oilphase, a suspension, a solid, a gel, a powder, a paste, a microneedle, afoam, or an aerosol composition. Compositions of such formulations maybe prepared according to the conventional methods in the pertinentfield.

The cosmetic composition according to the present specification mayfurther include functional additives and components included in generalcosmetic compositions in addition to the compounds, extracts orfractions of the present specification. The functional additive mayinclude the components selected from the group consisting ofwater-soluble vitamins, oil-soluble vitamins, polymer peptides, polymerpolysaccharides, sphingolipids and seaweed extracts. The cosmeticcomposition according to the present specification may include othercomponents that can give a synergistic effect to the main effect,preferably within a range that does not impair the main effect. Inaddition, the cosmetic composition according to the presentspecification may further include a moisturizer, an emollient, asurfactant, a ultraviolet absorbent, a preservative, a bactericide, anantioxidant, a pH adjusting agent, an organic and an inorganic pigment,a perfume, a cooling agent, or a control agent. The blending amount ofthe components may be easily selected by those skilled in the art withinthe range that does not impair the purpose and effect of the presentspecification, and the blending amount may be 0.001% to 10% by weight,specifically 0.01% to 3% by weight based on the total weight of thecomposition. have.

According to another embodiment, the composition may be a compositionfor external skin application. The composition for external skinapplication may be a composition such as cosmetics, mouthwashes,cleaning agents, pharmaceuticals and quasi-drugs, but is not limitedthereto. The external preparation for skin is not particularly limitedin its formulation.

In addition, the composition for external skin application of thepresent specification may further include at least one selected fromfunctional salts for a particular purpose and a pH adjusting agent foradjusting pH. In this case, the salt may be selected from inorganicsalts, organic salts and/or organic-inorganic salts for ion shielding,moisturizing, UV protection, or the like. For a concrete example, thesalt may be selected from sodium chloride (NaCl), sodium phosphate(Na3PO4), calcium chloride (CaCl₂)), or the like. The pH adjusting agentmay be selected from the group consisting of acids and bases, forexample hydrochloric acid, sulfuric acid, tartaric acid, citric acid,phosphoric acid, acetic acid, lactic acid, sodium lactate, sodiumhydroxide, potassium hydroxide, alkyl amines, alkanol amine and ammonia.

The composition for external skin application of the presentspecification may be a cosmetic, pharmaceutical or quasi-drugcomposition, wherein the cosmetic, pharmaceutical or quasi-drugcomposition may additionally contain a preservative, stabilizer,hydrating agents or emulsificant, adjuvant such as salt and/or bufferfor controlling osmotic pressure and other therapeutically usefulsubstances. The composition may be formulated into a lotion, cream,ointment, gel, or the like. The composition for external skinapplication may preferably be administered transdermally.

The dosage of the active ingredient of the pharmaceutical or quasi-drugcomposition may vary depending on the age, sex, and weight of thesubject to be treated, the specific disease or pathology to be treated,the severity of the disease or pathologic state, the route ofadministration, and the judgment of the prescriber. The determination ofdosage based on these factors is within the level of those skilled inthe art. In general, the dosage of the active ingredient may range from0.00001 mg/kg/day to 15 mg/kg/day, but is not limited thereto.

The pharmaceutical composition according to one aspect of the presentinvention may be administered orally, parenterally, rectally, topically,transdermally, intravenously, intramuscularly, intraperitoneally,subcutaneously, or the like. Formulations for oral administration maybe, but are not limited thereto, tablets, pills, soft and hard capsules,granules, powders, granules, solutions, emulsions or pellets.Formulations for parenteral administration may be, but are not limitedto, solutions, suspensions, liquids, emulsions, gels, injections, drops,suppositories, patches or sprays. The formulations can be readilyprepared according to conventional methods in the pertinent field, andmay additionally contain surfactants, excipients, hydrating agents,emulsificants, suspending agents, salts or buffers for controllingosmotic pressure, colorants, spices, stabilizers, preservatives,preserved agents or other commercially available adjuvants.

The dose or dosage of the pharmaceutical composition according to oneaspect of the present invention may vary depending on the age, sex,weight, pathologic state and severity of the subject to be administered,the route of administration or the judgment of the prescriber. Thedetermination of the dose based on these factors is within the level ofthose skilled in the art.

The formulation of the food composition is not particularly limited, butmay be, for example, formulated into tablets, granules, pills, powders,liquids such as drinks, caramels, gels, bars, tea bags, or the like. Inaddition to the active ingredient, the food composition of eachformulation may appropriately select and mix the ingredients that arecommonly used in the pertinent field according to the formulation orpurpose of use by those skilled in the art without difficulty.Synergistic effects may occur when applied simultaneously with other rawmaterials.

The composition may be administered by various methods such as simpleingestion, drinking, injection administration, spray administration orsqueeze administration, or the like.

Hereinafter, the configuration and effects of the present specificationwill be described in more detail with reference to the examples,experimental examples, and formulation examples. However, these examplesare provided only for the purpose of illustration in order to facilitateunderstanding of the present specification, and the scope and range ofthe present specification is not limited by the following examples.

EXAMPLES [Example 1] Preparation of Post Fermented Tea Samples

The water content was adjusted to 40% by weight by adding water to thegreen tea made from green tea (Camellia sinensis var. Yabukita) leaves.Then 5×10⁶ cfu/g of Bacillus subtillis was seeded, fermented at 50° C.for 3 days, and then fermented at 80° C. for 4 days.

The fermented tea sample was pulverized for 15 seconds and filteredthrough a stainless steel sieve of mesh size 1 mm. Then, 50 mg of thepulverized sample was added to 1.5 ml Eppendorf tube and 1 ml ofdeionized water was added and stirred at a constant speed for 30 minutesin a 60° C. constant temperature water bath, followed by centrifugationat 25° C., 13,000 rpm for 15 minutes. Only the portions insoluble inwater in the centrifuged fermented green tea extract were separated.

[Example 2] Obtainment of Fractions and Separation of Compounds

150 g of the post-fermented tea sample was fractionated with acetone toremove catechin derivatives and caffeine and to obtain a soluble inwhich other compounds were concentrated. For 40 g of the acetonesolubles, fractions were obtained primarily using a 5:1 (v/v) mixture ofchloroform:methanol as a solvent using silica gel column chromatography.

8.9 g of the caffeine-free chloroform:methanol 5:1 (v/v) fraction wasfractionated using large capacity high performance countercurrentchromatography (HPCCC, Dynamic Extractions Ltd, UK). The solvent usedthen was n-hexane-TBME (Methyl tert-butyl ether)-BuOH-MeCN-Water(0.25:3:1:1:5, v/v), and the flow rate was 25 ml/min. A total of 10subfractions were divided using the above conditions, and the componentscontained in each fraction was again separated using small capacityHPCCC (Dynamic Extractions Ltd, UK), High-performance liquidchromatography (HPLC), Sephadex LH-20 columns (GE HealthcareBio-Sciences, Sweden), or the like.

As a result,kaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside],a compound which was not known before, could be separated from thefractions. The structure of each compound was investigated byidentifying the structure using ¹H, ¹³C-NMR (nuclear magnetic resonancespectroscopy), UV (ultraviolet spectroscopy), and ESI-MS (Electro SprayIonization Mass Spectroscopy). In the case of ¹H and ¹³C nuclearmagnetic resonance (NMR), methanol-d3 was used as a solvent, and BrukerAdvance DPX-500 (BRUKER, USA) was used as a device. MS spectra of eachcompound were analyzed using 6200 Series Accurate-Mass Time-of-Flight(TOF) LC/MS (Agilent, US).

As a result of the analysis, each of the compound is a novel compoundwhich was not known before, and was confirmed askaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside](‘new material 33’) having a molecular weight of 902.2481 of C₄₂H₄₆O₂₂.

The formula and NMR data ofkaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside]are as follows.

TABLE 1 Position ¹³C-NMR ¹H-NMR 2 161.26 3 135.07 4 179.31 5 161.5 699.87 6.17 (H6, brs) 7 165.74 8 94.8 6.35 (H8, brs) 9 158.58 10  105.84 1′ 122.72 2′, 6′ 132.29 7.99 (H2′/H6′, d, J = 8.3 Hz) 3′, 5′ 116.276.87 (H3′/H5′, d, J = 8.3 Hz)  4′ 158.69 p-coumaric acid   1″′ 127.32″′, 6″′ 131.2 7.45 (H2″′/H6″′, d, J = 8.1 Hz) 3″′, 5″′ 116.82 6.80(H3″′/H5″′, d, J = 8.1 Hz)   4″′ 161.26   7″′ 115.31 6.35 (H7″′, d, J =15.7 Hz)   8″′ 146.88 7.67 (H8″′, d, J = 15.7 Hz) C═O 168.79 Glc1  1″101.55 5.46 (H1″, d, J = 7.8 Hz)  2″ 74.14 5.34 (H2″, t, J = 9 Hz)  3″73.25 3.76 (H3″, d, J = 10.4 Hz)  4″ 70.47 3.85 (H4″, m)  5″ 75.51 3.73(H5″, m)  6″ 67.54 3.76 (H6″, brd, J = 10.4 Hz) 3.54 (H6″, m) Rha   1″″101.85 4.60 (H1″″, brs)   2″″ 71.34 3.95 (2″″, m)   3″″ 83.09 3.61(H3″″, dd, J = 9, 3 Hz)   4″″ 72.6 3.46 (H4″″, m)   5″″ 69.49 3.54(H5″″, m)   6″″ 18.08 1.19 (H6″″, d, J = 6 Hz) Glc2  1″″′ 105.74 4.40(H1″″′, d, J = 7.5 Hz)  2″″′ 75.4 3.25 (H2″″′, m)  3″″′ 77.6 3.36(H3″″′, m)  4″″′ 70.84 3.36 (H4″″′, m)  5″″′ 77.6 3.25 (H5″″′, m)  6″″′62.05 3.71 (H6″″′, m)

The MS spectrum ofkaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside]is as shown in FIG. 1, ¹H-NMR spectrum and ¹³C-NMR spectrum are as shownin FIGS. 2 and 3, respectively, HSQC (Heteronuclear Single QuantumCoherence) spectrum is as shown in FIG. 4, and HMBC (HeteronuclearMultiple-Bond Coherence) spectrum is as shown in FIG. 5.

[Experimental Example 1] Evaluation of the TyrosinaseActivity-Inhibitory Effect

The tyrosinase activity-inhibitory effect of new material 33 wasevaluated.

Specifically, tyrosinase and tyrosine derived from mushrooms wereobtained from Sigma Chemical. Arbutin and new material 33 were treatedin different concentrations with 150 μl of 0.1 M phosphate buffer (pH6.5), 8 μl of mushroom tyrosinase (2,100 unit/ml), and 0.05 Mconcentration of L-tyrosine, respectively. Tyrosinase activity wasassessed by measuring the absorbance at 490 nm using a microplate reader(Bio-Rad 3550, Richnmond, Calif., U.S.A.) after an enzyme reaction for20 minutes at 37° C.

The tyrosinase activity-inhibitory effect (IC₅₀) by each new material 33was calculated and shown in Table 2 (the control group is a group nottreated with new material 33 and arbutin).

TABLE 2 Tyrosinase Activity-Inhibitory Effect of New Material Testmaterials IC₅₀ (ppm) Control — 33 11 Arbutin 149

[Experimental Example 2] Evaluation of the Melanin Production-InhibitoryEffect

The melanin production-inhibitory effect of new material 33 was measuredin comparison with Kojic acid, which is known to have an excellent skinwhitening effect.

Specifically, the degree of intracellular melanin production wasmeasured by Dooley's method. The cell line was a Mel-Ab cell line(Falcon, U.S.A.) derived from the skin pigment of C57BL/6. The culturewas performed under the condition of 37° C., 5% CO₂ in a DMEM mediumcontaining 10% fetal placental serum, 100 nM12-O-tetradecanoylphorbol-13-acetate, and 1 nM Cholera Toxin. Thecultured Mel-Ab cells were detached with 0.25% Trypsin-EDTA and seededagain into 24-well culture plates in the same number (1×10⁵ cells/well),and then the test materials were treated by replacing with a mediumcontaining the test materials for two consecutive days from the secondday. As test materials, 5 ppm, 10 ppm, 25 ppm and 50 ppm of new material33, 25 ppm, 50 ppm and 100 ppm of Kojic acid, and DMSO (control) wereused. After 5 days, melanin contained in cells was dissolved by treatingwith 1N-NaOH, and the amount of melanin was measured by measuringabsorbance at 400 nm. The concentration of materials (IC₅₀) needed toreduce melanin production in melanocytes by half was calculated and theresults are shown in Table 3 below.

TABLE 3 Melanin Production-Inhibitory Effect of New Materials Testmaterials IC₅₀ (ppm) Control — 33 15.7 Kojic acid 39.36

[Experimental Example 3] Skin Accumulation Stimulation Experiment

Human repeated insult patch tests (HRIPT) were performed to confirm theskin accumulation stimulation of thekaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside]and to calculate the concentration range available for the skin.

Specifically, 15 healthy adult subjects were randomly selected, and thetest composition (the skin composition including an emulsifier, astabilizer, purified water, etc. in addition to the compound) containing0.5 wt %, 1 wt %, and 3 wt % of the compound was added dropwise by 20 μlper chamber (IQ chamber, Epitest Ltd, Finland). After 24 hours frompatching the upper right part of the back of a subject, it was replacedwith the new patch. A total of 9 patches were conducted, three times aweek for total 3 weeks, in such manner, the skin reactions before andafter patch were examined every time, and the skin reactions until 48hours after removing the final patch were observed, and the averagereactivity was observed. The results are shown in Table 4 below.

TABLE 4 Test substance Number of subjects with ±, +, or ++ reactivityand (Unit: person) Average content 1st 2nd 3rd 4th 5th 6th 7th 8th 9threactivity Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0.5 wt % 0 0 0 0 0 0 0 0 0 0 of new 0 0 0 0 0 0 0 0 0 material 0 0 0 00 0 0 0 0 33 1 wt % of 0 0 0 0 0 0 0 0 0 0 new 0 0 0 0 0 0 0 0 0material 0 0 0 0 0 0 0 0 0 33 3 wt % of 0 0 0 0 0 0 0 0 0 0 new 0 0 0 00 0 0 0 0 material 0 0 0 0 0 0 0 0 0 33 [[Reactivity]] −: negative (noresponse) ±: doubtful or slight erythema, etc. +: weak reaction (whichis accompanied with no phlyctenule), erythema, papule ++: severereaction (which is accompanied with phlyctenule), erythema, papule,phlyctenule +++: strong reaction, bullae reaction [[Average reactivityequation]] Average Reactivity = [{(total sum of the values obtained bymultiplying the number of the examinees who exhibited the reactivity andthe reaction index)/(total number of the examinees × highest point (4point))} × 100]/number of examinations (9 tests). In the equation, ifthe reactivity is −, the reaction index is 0, if the reactivity is ±,the reaction index is 1, if the reactivity is +, the reaction index is2, and if the reactivity is ++, the reaction index is 4. When theaverage reactivity is less than 3, it is considered as a stablecomposition.

The skin response was determined according to the criteria of theInternational Contact Dermatitis Research Group (ICDRG). ‘New material33’ in the table above iskaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside].That is, all the substance showed (−) reactivity in the content range(no subject showed ±, +, ++, or +++ reactivity), through which it wasconfirmed that the substances have no cumulative irritation of the skinand can be used safely on the skin.

Hereinafter, formulation examples of the composition according to anaspect of the present disclosure will be explained, but the scope of thepresent disclosure is not limited thereto.

[Formulation Example 1] Soft Capsule

10 mg ofkaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside],80-140 mg of L-carnitine, 180 mg of soybean oil, 2 mg of palm oil, 8 mgof vegetable hardened oil, 4 mg of yellow wax and 6 mg of lecithin weremixed and filled in one capsule according to a conventional method toprepare a soft capsule.

[Formulation Example 2] Tablet

10 mg ofkaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside],200 mg of galactooligosaccharide, 60 mg of lactose, and 140 mg ofmaltose were mixed and granulated using a fluidized bed dryer, then 6 mgof sugar ester was added, and tableted with tablet machine to prepare atablet.

[Formulation Example 3] Granule

5 mg ofkaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside],250 mg of anhydrous crystalline glucose and 550 mg of starch were mixed,molded into granules using a fluidized bed granulator, and then filledinto pouch to prepare a granule.

[Formulation Example 4] Drink

2 mg ofkaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside],10 g of glucose, 0.6 g of citric acid, and 25 g of liquidoligosaccharides were mixed, and then 300 ml of purified water wasadded. Each bottle was filled with 200 ml. After the bottle was filled,they were sterilized at 130° C. for 4-5 seconds to prepare a drink.

[Formulation Example 5] Injection

20 mg ofkaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside],a suitable amount of sterile distilled water for injection, and asuitable amount of a pH adjusting agent were used to prepare aninjection in a conventional method.

[Formulation Example 6] Health food

The health food was prepared in a conventional method according to thecomposition shown in Table 5 below.

TABLE 5 Components Contents Kaempferol3-O-[2-O″-(E)-p-coumaroyl][beta-D-0.5 mg glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside] Vitamin mixture Vitamin A acetate 70 μgVitamin E 1.0 mg Vitamin B1 0.13 mg Vitamin B2 0.15 mg Vitamin B6 0.5 mgVitamin B12 0.2 μg Vitamin C 10 mg Biotin 10 μg Nicotinic acid amide 1.7mg Folic acid 50 μg calcium pantothenate 0.5 mg Mineral mixture ferroussulfate 1.75 mg zinc oxide 0.82 mg Magnesium carbonate 25.3 mg Potassiumdihydrogen phosphate 15 mg Dibasic calcium phosphate 55 mg Potassiumcitrate 90 mg Calcium carbonate 100 mg Magnesium chloride 24.8 mg

Although the composition ratio of the vitamin and inorganic mixture wasobtained by a mixed composition using the components that are relativelysuitable for health foods, the compounding ratio may be arbitrarilymodified. The above ingredients may be mixed according to theconventional method for preparing health foods, and then may be used forpreparing a health food composition according to the conventionalmethod.

[Formulation Example 7] Health drink

TABLE 6 Components Contents Kaempferol3-O-[2-O″-(E)-p-coumaroyl][beta-D-2 mg glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside] Citric acid 1000 mg Oligosaccharide 100 g Plumconcentrate 2 g Taurine 1 g Purified water balance Total volume 900 Ml

As shown in Table 6 above, the balance of purified water was added tomake a total volume of 900 ml, and the above components were mixedaccording to the conventional method for preparing a healthy beverage.The mixture was stirred and heated at 85° C. for about 1 hour, and thenthe resulting solution was filtered, obtained in a sterilized 2 litercontainer, sterilized and sealed, and then refrigerated to prepare ahealth drink.

[Formulation Example 8] Softening tonic (Skin Lotion)

0.2 wt % ofkaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside],1.00 wt % of L-ascorbic acid-2-magnesium phosphate salt, 5.00 wt % ofwater-soluble collagen (1% aqueous solution), 0.10 wt % of sodiumcitrate, 0.05 wt % of citric acid, 0.20 wt % of licorice extract, 3.00wt % of 1,3-butylene glycol, and the residual quantity of purified waterwere used to prepare softening tonic (skin lotion).

[Formulation Example 9] Cream Formulation

0.2 wt % ofkaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside],2.00 wt % of polyethyleneglycol monostearate, 5.00 wt % ofself-emulsifying glycerin monostearate, 4.00 wt % of propylene glycol,6.00 wt % of squalene, 6.00 wt % of tri2-ethylhexaneglyceryl, 1.00 wt %of sphingoglycolipid, 7.00 wt % of 1,3-butylene glycol, 5.00 wt % ofbeeswax, and the balance of purified water were used to prepare a creampreparation.

[Formulation Example 10] Pack

0.2 wt % ofkaempfero13-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside],21.00 wt % of polyvinyl alcohol, 3.00 wt % of L-ascorbicacid-2-magnesium phosphate, 5.00 wt % of lauroylhydroxyproline, 8.00 wt% of water-soluble collagen (1% aqueous solution), 7.00 wt % of1,3-butylene glycol, 7.00 wt % of ethanol, and the balance of purifiedwater were used to prepare a composition and then to prepare a pack.

From the foregoing, the present disclosure has been described withreference to the specific embodiments of the present specification, andit is apparent to those skilled in the art that these specifictechniques are only preferred embodiments, which are not intended tolimit the scope of the present specification. Accordingly, thesubstantial scope of the present specification will be defined by theappended claims and equivalents thereof.

1. A method for skin whitening comprising administering a compound offollowing Formula 1, an optical isomer thereof, a pharmaceuticallyacceptable salt thereof, a hydrate thereof, a solvate thereof, or apost-fermented tea extract comprising the same to a subject in needthereof:

(In Formula 1 above, R₁ is C₁₅H₉O₆, R₂ is C₆H₁₁O₅, and R₃ is C₉H₇O₂). 2.The method of claim 1, wherein the R₁ is represented by followingFormula 2:


3. The method of claim 1, wherein the R₂ is represented by followingFormula 3:


4. The method of claim 1, wherein the R₃ is represented by followingFormula 4:


5. The method of claim 1, wherein the compound iskaempferol3-O-[2-O″-(E)-p-coumaroyl][beta-D-glucopyranosyl-(1→3)-O-alpha-L-rhamnopyranosyl-(1→6)-O-beta-D-glucopyranoside].6. The method of claim 1, wherein the compound of following Formula 1,the optical isomer thereof, the pharmaceutically acceptable saltthereof, the hydrate thereof, the solvate thereof, or the post-fermentedtea extract comprising the same inhibits one or more of tyrosinaseactivity and melanin production.
 7. The method of claim 1, wherein thecompound of following Formula 1, the optical isomer thereof, thepharmaceutically acceptable salt thereof, the hydrate thereof, thesolvate thereof, or the post-fermented tea extract comprising the sameprevents, improves or treats one or more selected from the groupconsisting of spots, freckles, lentigos, birthmarks, melanomas,ultraviolet radiation-induced pigmentation, drug-induced pigmentation,pigmentation following inflammation and pigmentation caused bydermatitis.
 8. The method of claim 1, wherein the extraction is anextraction by one or more solvents selected from hydrothermal water,lower alcohols of C₁ to C₆, and mixed solvents thereof.
 9. The method ofclaim 8, wherein the lower alcohol is ethanol.
 10. The method of claim1, wherein the extract is a fraction fractionated with ketones afterextraction
 11. The method of claim 10, wherein the ketone is acetone.12. The method of claim 1, wherein the compound of Formula 1, theoptical isomer thereof, the pharmaceutically acceptable salt thereof,the hydrate thereof, or the solvate thereof is administered in form of acomposition, and wherein the content of the compound of Formula 1, theoptical isomer thereof, the pharmaceutically acceptable salt thereof,the hydrate thereof, or the solvate thereof in the composition is0.00001% to 10% by weight based on the total weight of the composition.13. The method of claim 1, wherein the post-fermented tea extract isadministered in form of a composition, and wherein the content of thepost-fermented tea extract in the composition is 0.1% to 90% by weightbased on the total weight of the composition.
 14. The method of claim 1,wherein the extract comprises the compound of Formula 1, the opticalisomer thereof, the pharmaceutically acceptable salt thereof, thehydrate thereof, or the solvate thereof in an amount of 0.00001% to 20%by weight based on the total weight of the extract.
 15. The method ofclaim 1, wherein the dosage of the compound of Formula 1, the isomerthereof, the pharmaceutically acceptable salt thereof, the hydratethereof, or the solvate thereof is 0.00001 mg/kg/day to 100 mg/kg/day.16. The method of claim 1, wherein the compound of following Formula 1,the optical isomer thereof, the pharmaceutically acceptable saltthereof, the hydrate thereof, the solvate thereof, or the post-fermentedtea extract comprising the same is administered in form of acomposition, and wherein the composition is a food composition, acosmetic composition, or a pharmaceutical composition.